Test piece for assaying high density lipoprotein (hdl) cholesterol

ABSTRACT

A test piece having a simple structure by which HDL cholesterol can be easily assayed by using a small amount of a specimen. A reagent layer ( 2 ) is formed on a support ( 1 ). This reagent layer ( 2 ) contains an enzyme reagent for assaying cholesterol, a first surfactant capable of highly solubilizing HDL (high density lipoprotein) compared with lipoproteins other than HDL, and a second surfactant inhibiting the dissolution of lipoproteins other than HDL.

TECHNICAL FIELD

[0001] The present invention relates to a test piece for measuringhigh-density lipoprotein (HDL) cholesterol.

BACKGROUND ART

[0002] HDL cholesterol is one of the important items to be measured inmedical examinations, various tests for clinical medicine, and the like,from the epidemiological viewpoint that the HDL cholesterol iscorrelated inversely with the frequency of the occurrence of coronarydiseases. On the other hand, in clinical tests and the like, test piecesproduced by impregnating filter paper or the like with a reagent andthen drying it have been used widely because it is necessary to treat alarge number of specimens.

[0003] One example of a method for measuring HDL cholesterol isseparating HDL and other lipoproteins (such as LDL and VLDL) byultracentrifugation, and then measuring only HDL cholesterol using anenzyme. Another example of the method is separating HDL and otherlipoproteins by electrophoresis and staining the lipid to measure theintensity of the color developed. Although they are general methods formeasuring HDL cholesterol, they are not practiced in the clinical testsor the like because they require a complicated operation, which makes itdifficult to treat a large number of specimens. On this account, avariety of methods for measuring HDL cholesterol that enable thetreatment of a large number of specimens have been proposed and arepracticed actually in some clinical tests and the like. Examples of suchmethods will be described in the following.

[0004] For example, there have been proposed chemically modifyingcholesterol esterase and cholesterol oxidase and specifically reactingthe cholesterol in HDL in the presence of an inclusion compound such ascyclodextrin (JP 7(1995)-301636 A); adding an aggregating agent to aspecimen to aggregate and settle lipoproteins other than HDL and thenmeasuring HDL cholesterol present in the supernatant (for example, JP8(1996)-131197 A, Japanese Patent No. 2600065, JP 8(1996)-201393 A, andJapanese Patent No. 2799835); and using a surfactant to generate a timeperiod in which an enzyme reacts with HDL cholesterol specifically andmeasuring the HDL cholesterol in this time period (JP 11(1999)-56395 A).However, these methods are measurement methods applicable to a liquidsystem and cannot be applied directly to a test piece, which is ameasurement method employing a dry system.

[0005] Examples of a method for measuring HDL cholesterol using a testpiece include a method utilizing the above-described aggregationreaction (JP 2(1990)-210265 A, JP 3(1991)-99268 A, etc.). However, thismethod has a drawback in that it requires a filter separation mechanismor the like so that the structure of the test piece becomes complicatedand a large amount of specimen is required.

DISCLOSURE OF INVENTION

[0006] The present invention has been made in view of theabove-described circumstances. It is an object of the present inventionto provide a test piece having a simple structure, by which HDLcholesterol can be measured easily using a small amount of specimen.

[0007] In order to achieve the above-described object, a test piece formeasuring HDL cholesterol according to the present invention includes:an enzyme reagent for measuring cholesterol; a first surfactant thatmakes a solubility of HDL higher than that of a lipoprotein other thanthe HDL; and a second surfactant that inhibits the lipoprotein otherthan the HDL from dissolving.

[0008] As described above, by letting the two types of surfactants becontained in the test piece of the present invention, the enzyme reagentfor measuring cholesterol can measure the HDL cholesterol specificallyand easily. Further, this test piece does not utilize any specialreaction such as an aggregation reaction and thus does not require anyspecial structure. Therefore, the test piece of the present inventioncan have a simple structure and requires only a small amount ofspecimen.

[0009] The test piece according to the present invention may have astructure in which a reagent layer is formed on a support, and theenzyme reagent, the first surfactant, and the second surfactant arecontained in the reagent layer. In this case, the reagent layer mayinclude a sample supply layer and a detecting layer. The firstsurfactant and the second surfactant may be contained in the samplesupply layer and the enzyme reagent may be contained in the detectinglayer. Further, it is preferable that, inside the sample supply layer,the second surfactant is contained in an upper portion (an upper layer)and the first surfactant is contained in a lower portion (a lowerlayer). Furthermore, it is also preferable that the first surfactant,the second surfactant, and a part of the enzyme reagent are contained inthe sample supply layer, and the rest of the enzyme reagent is containedin the detecting layer.

[0010] As the first reagent, polyoxyethylene alkylene phenyl ether andpolyoxyethylene alkylene tribenzyl phenyl ether are preferable, whichmay be used alone or in combination. Of these two surfactants,polyoxyethylene alkylene tribenzyl phenyl ether is more preferable.

[0011] As the second reagent, polyoxyethylene alkylether,polyoxyethylene alkyl phenyl ether, a polyoxyethylene-polyoxypropylenecondensation product, polyoxyethylene alkylether sulfate, and alkylbenzenesulphonate are preferable, which may be used alone or in acombination of two or more types. Among these, thepolyoxyethylene-polyoxypropylene condensation product is the mostpreferable.

[0012] As the enzyme reagent, a combination of cholesterol esterase andcholesterol oxidase and a combination of cholesterol esterase andcholesterol dehydrogenase are preferable. Of these two combinations, theformer is more preferable. After the sample is added and mixed with thereagents, the proportion of the cholesterol esterase is, for example, inthe range from 5 to 1000 U/mL, preferably from 10 to 100 U/mL, and morepreferably from 30 to 70 U/mL. The proportion of the cholesterol oxidasein all the components during the enzyme reaction is, for example, in therange from 5 to 1000 U/mL, preferably from 10 to 100 U/mL, and morepreferably from 20 to 50 U/mL. The proportion of the cholesteroldehydrogenase in all the components during the enzyme reaction is, forexample, in the range from 5 to 1000 U/mL, preferably from 10 to 100U/mL, and more preferably from 30 to 70 U/mL. As the enzymes,commercially available enzymes can be used. However, considering theprinciples of reaction in the present invention, it is preferable to usepure products in which a contaminant that interferes with the reactionsteps (e.g., a substance that might degrade the characteristics of thefirst surfactant or the second surfactant) is not present, e.g., generecombination products and the like. In the present invention, theproportion in all the components during the enzyme reaction refers tothe proportion in all the components after the specimen (sample) hasbeen added, and all the components refers to the components presentduring the enzyme reaction altogether, such as the sample, the enzymereagent, the first surfactant, and the second surfactant. Generally, theamount of one specimen can be predicted or determined from the typethereof.

[0013] The proportion of the first surfactant in all the componentsduring the enzyme reaction preferably is in the range from 1 to 10 wt %,more preferably from 3 to 7 wt %. On the other hand, the proportion ofthe second surfactant in all the components during the enzyme reactionpreferably is in a range from 1 to 20 wt %, more preferably from 7 to 13wt %. It is to be noted here that the proportion of these surfactants isconsiderably higher than that in the case where a liquid system isemployed. The weight ratio (A/B) of the first surfactant (A) to thesecond surfactant (B) preferably is in the range from 1/10 to 1/1, morepreferably from 1/3 to 2/3. Further, it is preferable that the reactionpH at the test piece according to the present invention is adjusted tobe in the range from 6 to 9. The pH in the range from 7 to 8 is morepreferable, and the optimal pH is 7.7.

[0014] A specimen to be measured with the test piece according to thepresent invention in not specifically limited, and may be any specimens(especially biological liquids) that may contain HDL cholesterol, suchas whole blood, plasma, and serum, for example.

BRIEF DESCRIPTION OF DRAWINGS

[0015]FIG. 1(A) is a cross-sectional view showing a structure of a testpiece according to one example of the present invention; FIG. 1(B) is across-sectional view showing a structure of a test piece according toanother example of the present invention; and FIG. 1(C) is across-sectional view showing a structure of a test piece according tostill another example of the present invention.

[0016]FIG. 2 is a graph showing the change in reflectance overtime in atest piece according to still another example of the present invention.

[0017]FIG. 3 is a graph showing a correlation between the measured valueof HDL cholesterol obtained by a test piece according to still anotherexample of the present invention and the measured value of the sameobtained by the aggregation method.

[0018]FIG. 4 is a graph showing a correlation between the measured valueof HDL cholesterol obtained by a test piece according to still anotherexample of the present invention and the measured value of the sameobtained by the direct method.

BEST MODE FOR CARRYING OUT THE INVENTION

[0019] Hereinafter, a test piece of the present invention will bedescribed by way of examples.

[0020] First, as an enzyme reagent for measuring cholesterol to be usedin the present invention, the above-described two combinations can beused, for example, and the combination of cholesterol esterase andcholesterol oxidase preferably is used. HDL cholesterol in vivogenerally is divided into two types depending on its state, i.e., a freestate and the state where it is bound to a fatty acid by an esterlinkage. Thus, the HDL cholesterol in the latter state is degradedhydrolytically with the cholesterol esterase to generate cholesterol andthe fatty acid. Thereafter, the cholesterol thus generated is treatedwith the cholesterol oxidase to generate cholestenone and hydrogenperoxide. Then, by measuring the hydrogen peroxide, the amount of theHDL cholesterol can be obtained. The measurement of the hydrogenperoxide can be carried out by an electrochemical measuring method or achemical measuring method. However, a chemical measuring method using aperoxidase (POD) and a substrate that develops color by oxidation(hereinafter, referred to as “oxidation color-developing substrate”)preferably is used. In this case, the degree of color development of theoxidation color-developing substrate can be measured optically. Thus, inthis case, the enzyme reagent includes the POD and the oxidationcolor-developing substrate. The proportion of the POD in all thecomponents during the enzyme reaction is in the range from 50 to 5000U/mL, preferably from 100 to 1500 U/mL, and more preferably from 700 to800 U/mL, for example. The proportion of the oxidation color-developingsubstrate in all the components during the enzyme reaction may bedetermined as appropriate, depending on the types thereof or the like.However, for example, the proportion is in the range from 10 to 1000mmol/L, preferably from 20 to 200 mmol/L, and more preferably from 30 to70 mmol/L.

[0021] Examples of the oxidation color-developing substrate include:ALPS [N-Ethyl-N-(3-sulfopropyl) aniline sodium salt]; DAOS[N-Ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline sodium salt];MAOS [N-Ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethylaniline sodiumsalt monohydrate]; TOOS[N-Ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline, sodium saltdihydrate]; and phenols. Among these, the substrate that develops colormeasurable at a wavelength used by a measuring apparatus can be selectedas appropriate.

[0022] The first surfactant is not specifically limited, as long as itcan make the solubility of HDL higher than that of other lipoproteins(such as LDL and VLDL). The first surfactant has a function ofsolubilizing cholesterol or cholesterol esters contained in the HDL,which brings about the effect that the reaction between the cholesterolor esters thereof and the enzyme reagent is accelerated. As the firstsurfactant, commercially available products may be used. An example of acommercially available polyoxyethylene alkylene phenyl ether is aproduct named “EMULGEN A-66” (Kao Corporation), and an example of acommercially available polyoxyethylene alkylene tribenzyl phenyl etheris a product named “EMULGEN B-66” (Kao Corporation).

[0023] The second surfactant is not specifically limited, as long as itcan inhibit the lipoproteins other than the HDL (such as LDL and VLDL)from dissolving. By inhibiting the dissolution of the lipoproteins otherthan the HDL by the second surfactant, the reaction between cholesterolor esters thereof contained in the lipoproteins other than the HDL andthe enzyme reagent is inhibited. As the second surfactant, theabove-described surfactants preferably are used. For example,polyoxyethylene cetyl ether (e.g., a product named “EMULGEN 220”available from Kao Corporation) preferably is used as polyoxyethylenealkylether; polyoxyethylene nonyl phenyl ether (e.g., a product named“EMULGEN 913” available from Kao Corporation) preferably is used aspolyoxyethylene alkyl phenyl ether; a product named “Pluronic F-88”available from Asahi Denka Co., Ltd. preferably is used as apolyoxyethylene-polyoxypropylene condensation product; sodiumpolyoxyethylene lauryl ether sulfate (e.g., a product named “EMAL 20C”available from Kao Corporation) preferably is used as polyoxyethylenealkylether sulfate; and sodium dodecylbenzenesulfonate preferably isused as alkyl benzenesulphonate. They can be used alone or in acombination of two or more types.

[0024] By using the first surfactant and the second surfactant incombination as described above, a time period is generated in which theenzyme reagent specifically reacts with HDL cholesterol. By carrying outthe measurement in this time period, it becomes possible to measure theHDL cholesterol specifically. This time period is, for example, from 1minute after the start of the reaction (i.e., after adding the specimen)to 20 minutes after the start of the reaction, preferably from 3 minutesafter the start of the reaction to 15 minutes after the start of thereaction, and more preferably from 5 minutes after the start of thereaction to 10 minutes after the start of the reaction. The test pieceaccording to the present invention may include, in addition to theenzyme reagent, the first surfactant, and the second surfactant, otheradditives such as an antioxidant, an anti-reduction agent, and a reagentfor removing an interfering substance, if necessary. Such additives maybe contained in the test piece in a proportion causing no damage to thefunction of the enzyme reagent, the first surfactant, and the secondsurfactant.

[0025] Next, one example of a structure of a test piece according to thepresent invention is shown in the cross-sectional view of FIG. 1.

[0026] In a test piece shown in FIG. 1(A), a reagent layer 2 is formedon a support 1. The above-described enzyme reagent, first surfactant,and second surfactant are contained in this reagent layer 2. The support1 may be made of a resin such as polyethylene terephthalate (PET),polystyrene, polyester, or cellulose acetate. Among these, PET ispreferable. The thickness of the support 1 is not specifically limited,and can be, for example, in the range from 0.1 to 1 mm, preferably from0.1 to 0.5 mm, and more preferably from 0.2 to 0.3 mm. The reagent layer2 can be formed, for example, by dissolving the enzyme reagent, thefirst surfactant, the second surfactant, and a hydrophilic polymer in asolvent, applying the resultant mixture onto the support 1 and thendrying it. Examples of the hydrophilic polymer includepolyvinylpyrrolidone, hydroxypropylcellulose, methylcellulose, sodiumalginate, polyacrylic acid, gelatin, acid-hydrolytically-degradedgelatin, polyacrylamide, and agarose. Examples of the solvent include aphosphate buffer, Good's buffers, and a Tris-HCl buffer. Among these,the Good's buffers are preferable. As the Good's buffers, PIPES, TES,HEPES, DIPSO, TAPSO, and MES are preferable, and TES are morepreferable. The pH of the buffer is adjusted preferably to be in therange from 6 to 9, more preferably from 7 to 8, and optimally to be 7.7.The proportion of the hydrophilic polymer is, for example, in the rangefrom 0 to 40 wt %, more preferably from 10 to 30 wt % of the totalweight of the solution to be applied onto the support 1, and can beadjusted as appropriate, depending on the type thereof. The reagentssuch as the enzyme reagent, the first surfactant, and the secondsurfactant are adjusted before being added to the solvent so that theyare contained in the resultant solution in the above-describedproportions. Further, the method for applying the solution is notspecifically limited, and can be, for example, a method using a brush orthe like, spray coating, a method using a roll coater, and dipping. Thethickness of the reagent layer 2 is, for example, in the range from 50to 250 μm, preferably from 80 to 200 μm, and more preferably from 100 to150 μm. The reagent layer 2 may contain the above-described additivessuch as an antioxidant, if necessary, in addition to the above-describedcomponents. The shape of the test piece is not specifically limited.However, the test piece generally has a plate-like shape or a stripshape. In the case where the test piece has a strip shape, the size ofthe test piece is, for example, in the range from 50 to 150 mm for totallength and in the range from 30 to 15 mm for width, and preferably inthe range from 60 to 100 mm for total length and in the range from 5 to10 mm for width.

[0027] The test piece can be used in the following manner, for example.First, a specimen such as blood is dripped on the reagent layer 2. Thespecimen dripped reacts with the reagents contained in the reagent layer2, and a time period in which the enzyme reagent specifically reactswith HDL cholesterol is generated by the action of the first surfactantand the second surfactant. Then, after the start of the reaction (i.e.,after dripping the specimen), the HDL cholesterol is measured in theabove-described time period. When the reagent layer 2 contains POD andthe oxidation color-developing substrate, the color development of thesubstrate is measured with an optical measuring apparatus such as aspectrophotometer or a reflectometer. In the case where the sample isplasma or serum, the color development can be measured in the followingmanner. When the support 1 is optically transparent, the test piece isirradiated with light from the support 1 side or the reagent layer 2side and the transmitted light or the reflected light is measured. Onthe other hand, when the support is not optically transparent, it ispreferable that the test piece is irradiated with light from the reagentlayer 2 side and a reflectance or the like is measured. Further, in thecase where the sample is whole blood as will be described later, thecolor of red blood cells interferes with the measurement of light. Onthis account, it is preferable to provide a known blood-cell shieldinglayer and light-reflecting layer between the detecting layer and thesample supply layer, use an optically transparent support, and carry outthe measurement from this support side.

[0028] Next, in a test piece shown in FIG. 1(B), a detecting layer 2 bis formed on a support 1, and a sample supply layer 2 a is formed on thedetecting layer 2 b. A reagent layer 2 is formed of the detecting layer2 b and the sample supply layer 2 a. The first surfactant and the secondsurfactant are contained in the sample supply layer 2 a, and the enzymereagent is contained in the detecting layer 2 b. The reagent layer 2 canbe formed in the following manner, for example. First, the detectinglayer 2 b is formed by applying a solution prepared by dissolving theenzyme reagent and a hydrophilic polymer in a solvent onto the support 1and drying it. On the other hand, a porous member is impregnated with asolution in which the first surfactant and the second surfactant aredissolved and dried. The porous member serves as the sample supply layer2 a after being laminated on the detecting layer 2 b, whereby thereagent layer 2 is formed. In this case, the type and the concentrationof the hydrophilic polymer and the solvent are as described above.Further, as described above, the sample supply layer 2 a preferably isporous. As the porous member, knit fabric can be used, for example.Examples of the knit fabric include polyethylene knit fabric, polyesterknit fabric, polytetrafluoroethylene knit fabric, polysulfone knitfabric, polypropylene knit fabric, polyvinylidene difluoride knitfabric, cellulose mixed ester knit fabric, and glass fiber knit fabric.The thickness of the sample supply layer 2 a is, for example, in therange from 50 to 400 μm, preferably from 200 to 300 μm, and morepreferably from 220 to 270 μm. The thickness of the detecting layer 2 bis, for example, in the range from 5 to 100 μm, preferably from 5 to 50μm, and more preferably from 5 to 20 μm. This test piece can be used inthe same manner as in the above-described example. In this test piece, aspecimen contacts with the enzyme reagent after having contacted withthe first surfactant and the second surfactant, which allows HDLcholesterol to be measured more specifically. Further, as describedabove, it is preferable that the sample supply layer contains theabove-described two types of the surfactants and a part of the enzymereagent, and the detecting layer contains the rest of the enzymereagent. For example, cholesterol esterase, cholesterol oxidase, thefirst surfactant, and the second surfactant may be contained in thesample supply layer and the rest of the enzyme reagent, such as POD andthe oxidation color-developing substrate, may be contained in thedetecting layer.

[0029] In the test piece of the example shown in FIG. 1(B), the samplesupply layer 2 a may include an upper layer and a lower layer inside,and the second surfactant may be contained in the upper layer and thefirst surfactant may be contained in the lower layer. FIG. 1(C) shows atest piece according to this example. In FIG. 1(C), numeral 21 denotesan upper layer of a sample supply layer 2 a, and numeral 22 denotes alower layer of the sample supply layer 2 a. Other than these, componentsin common between FIG. 1(B) and FIG. 1(C) are numbered identically.According to this structure, a specimen first contacts with the secondsurfactant, then contacts with the first surfactant, and finallycontacts with the enzyme reagent. This allows HDL cholesterol to bemeasured still more specifically. The sample supply layer 2 a with thisstructure can be formed in the following manner, for example. After adetecting layer 2 b is formed in the above-described manner, a solutionprepared by dissolving the first surfactant and a hydrophilic polymer ina solvent is applied onto the detecting layer 2 b and dried, andthereafter, a solution prepared by dissolving the second surfactant anda hydrophilic polymer in a solvent further is applied thereto and dried.In this case, the type and the concentration of the hydrophilic polymerand the solvent are as described above, and the method for applying thesolutions is also as described above. The method for forming the testpiece shown in FIG. 1(C) is not limited to applying the solutionssuccessively, and may be as follows. First, the lower layer is formed byimpregnating a porous member with the first surfactant. Then, the upperlayer is formed by applying the second surfactant onto one surface ofthe porous member. Thereafter, the porous member is laminated on thedetecting layer 2 b so that the lower layer is in contact with thedetecting layer 2 b, thereby forming the test piece shown in FIG. 1(C).

[0030] Further, if necessary, a test piece according to the presentinvention further may include a blood-cell separating layer on a reagentlayer. As the blood-cell separating layer, a porous member capable offiltering blood cells may be provided, and in some cases, a knownreflecting layer may be provided along with the porous member. As theporous member, a known blood-cell separating material such as a glassfilter can be used, for example. According to the test piece providedwith the blood-cell separating layer as described above, HDL cholesterolin serum or plasma can be measured by supplying whole blood as it is.

EXAMPLES

[0031] Hereinafter, examples of the present invention will be describedalong with comparative examples. In the following description, a “K/S”value refers to a variable obtained by the following equation. The K/Svalue is used when converting a reflectance (R) into a concentration.

K/S=(1−R)²/2R

[0032] R: reflectance

Example 1

[0033] A solution for forming a detecting layer, having the followingcomposition, was applied onto a white PET film (125 μm thickness) so asto be 150 μm in thickness and then dried at 40° C. for 10 minutes. Thus,a detecting layer was formed. On the other hand, a polyethylene knitfabric (250 μm thickness) was impregnated with an impregnating solutionhaving the following composition, and then dried at 40° C. for 10minutes. After that, a coating solution was applied onto one surface ofthe polyethylene knit fabric and then dried at 40° C. for 10 minutes.Thus, a sample supply layer was formed. Subsequently, distilled waterwas sprayed over the surface of the detecting layer, and the samplesupply layer was laminated on the surface of the detecting layer so thatthe surface of the sample supply layer opposite to the surface ontowhich the coating solution had been applied was in contact with thesurface of the detecting layer. The thus-obtained laminate was thendried at 40° C. for 10 minutes. Thus, a desired test piece was produced.This test piece had a structure as shown in FIG. 1(C). Further,compositions of the respective solutions shown in the following indicatea final concentration (i.e., the concentration during the enzymereaction) when 5 μL of a sample was supplied. The reaction pH at thistest piece during the enzyme reaction was 7.7. (Composition ofImpregnating Solution) TES buffer (Dojindo Laboratories, pH 7.7) 30mmol/L cholesterol esterase 39 U/mL cholesterol oxidase 23 U/mLpolyoxyethylene alkylene tribenzyl phenyl ether (Kao Corporation,product name EMULGEN B66) 5 wt % (Composition of Coating Solution)polyoxyethylene-polyoxypropylene condensation product 10 wt % (AsahiDenka Co., Ltd., product name Pluronic F-88) (Composition of Solutionfor Forming Detecting Layer) POD (Toyobo Co., Ltd.) 693 U/mL4-aminoantipyrine (Kishida Chemical Co., Ltd.) 43 mmol/L DAOS (DojindoLaboratories) 51 mmol/L TES buffer (Dojindo Laboratories, pH 7.7) 300mmol/L

Comparative Example 1 and Comparative Example 2

[0034] A test piece was produced in the same manner as in Example 1except that neither polyoxyethylene alkylene tribenzyl phenyl ether (thefirst surfactant) nor the polyoxyethylene-polyoxypropylene condensationproduct (the second surfactant) was used (Comparative Example 1).Further, a test piece was produced in the same manner as in Example 1except that only the first surfactant was used (the second surfactantwas not used) (Comparative Example 2).

[0035] With regard to the test pieces of Example 1, Comparative Example1, and Comparative Example 2 produced in the manner described above, areflectance (time course) was measured with a special-purposereflectometer (ARKRAY, INC., product name SPOTCHEM) using serum as asample. The measurement wavelength was 610 nm. The results are shown inthe graph of FIG. 2. In this graph, the horizontal axis indicates ameasurement time (seconds) and the vertical axis indicates a K/S valuedescribed above.

[0036] As apparent from the graph of FIG. 2, according to the test pieceof Example 1 in which the first surfactant and the second surfactantwere used in combination, the reaction terminated in a short time andonly the HDL cholesterol could be measured. In contrast, according tothe test piece of Comparative Example 1 in which neither of theabove-described two surfactants was used, the reaction was weak and themeasurement of the HDL cholesterol was substantially impossible. Also,according to the test piece of Comparative Example 2 in which only thefirst surfactant was used, the HDL cholesterol could not be measuredspecifically.

Example 2

[0037] Three types of test pieces (A, B, C) were produced in the samemanner as in Example 1 except that the final concentrations of the firstsurfactant and the second surfactant were adjusted as shown in Table 1below. With regard to 60 serum samples with a known HDL cholesterolconcentration, a reflectance was measured in the same manner as inExample 1 using these three test pieces. Then, from the reflectance thusmeasured, the amount of the HDL cholesterol was calculated using theabove-described “K/S” value. Then, the correlation between thethus-obtained concentration and the known concentration was examined.The results also are shown in Table 1 below. TABLE 1 1st 2nd correlationsurfactant surfactant coefficient (r) test piece A 1 wt % 1 wt % 0.87test piece B 2 wt % 2 wt % 0.94 test piece C 4 wt % 4 wt % 0.97

[0038] As shown in Table 1, all of these three test pieces A, B, and Cexhibited a high correlation coefficient.

Example 3

[0039] The same test piece as than of Example 1 was produced. HDLcholesterol in 60 serum samples was measured in the same manner as inExample 2 using this test piece. HDL cholesterol in these samples wasmeasured also by the aggregation method and the direct method asdescribed in the following. Thereafter, the correlations between themeasured value obtained by the test piece and that obtained by theaggregation method and between the measured value obtained by the testpiece and that obtained by the direct method were examined. The graph ofFIG. 3 shows the correlation between the measured value obtained by thetest piece and that obtained by the aggregation method. The graph ofFIG. 4 shows the correlation between the measured value obtained by thetest piece and that obtained by the direct method.

[0040] (Aggregation Method)

[0041] Using a commercially available kit for measuring HDL cholesterolin serum (Eiken Chemical Co., Ltd., product name HDL-C55), themeasurement was carried out in accordance with directions for usethereof. After the reagents and the specimen were mixed with each other,the mixture was centrifuged at 3000 rpm for 10 minutes.

[0042] (Direct Method)

[0043] Using a commercially available kit for measuring HDL cholesterolin serum (Daiichi Pure Chemical Co., Ltd., product name Cholestest HDL),the measurement was carried out in accordance with directions for usethereof.

[0044] As shown in the graph of FIG. 3, there was a strong correlationbetween the measured value obtained by the test piece and that obtainedby the aggregation method. The correlation coefficient was r=0.962.Similarly, as shown in the graph of FIG. 4, there was a strongcorrelation between the measured value obtained by the test piece andthat obtained by the direct method. The correlation coefficient wasr=0.974.

[0045] INDUSTRIAL APPICABILITY

[0046] As specifically described above, a test piece according to thepresent invention has a simple structure and can measure HDL cholesteroleasily using a small amount of specimen. Therefore, the test piece ofthe present invention makes it possible to treat a large number ofspecimens in a short time and thus contributes to improvement inefficiency of clinical tests, in particular.

1. A test piece for measuring high-density lipoprotein (HDL) cholesterolcomprising: an enzyme reagent for measuring cholesterol; a firstsurfactant that makes a solubility of HDL higher than that of alipoprotein other than the HDL; and a second surfactant that inhibitsthe lipoprotein other than the HDL from dissolving.
 2. The test pieceaccording to claim 1, wherein a reagent layer is formed on a support,and the enzyme reagent, the first surfactant, and the second surfactantare contained in the reagent layer.
 3. The test piece according to claim2, wherein the reagent layer includes a sample supply layer and adetecting layer, the first surfactant and the second surfactant arecontained in the sample supply layer, and the enzyme reagent iscontained in the detecting layer.
 4. The test piece according to claim3, wherein the second surfactant is contained in an upper portion of thesample supply layer and the first surfactant is contained in a lowerportion of the sample supply layer.
 5. The test piece according to claim3, wherein the first surfactant, the second surfactant, and a part ofthe enzyme reagent are contained in the sample supply layer, and therest of the enzyme reagent is contained in the detecting layer.
 6. Thetest piece according to claim 1, wherein the first surfactant is atleast one surfactant selected from polyoxyethylene alkylene phenyl etherand polyoxyethylene alkylene tribenzyl phenyl ether.
 7. The test pieceaccording to claim 1, wherein the second surfactant is at least onesurfactant selected from the group consisting of polyoxyethylenealkylether, polyoxyethylene alkyl phenyl ether, apolyoxyethylene-polyoxypropylene condensation product, polyoxyethylenealkylether sulfate, and alkyl benzenesulphonate.
 8. The test pieceaccording to claim 1, wherein the enzyme reagent contains cholesterolelastase and cholesterol oxidase.
 9. The test piece according to claim1, wherein the enzyme reagent contains cholesterol elastase andcholesterol dehydrogenase.
 10. The test piece according to claim 1,wherein a proportion of the first surfactant in all the componentsduring an enzyme reaction is in a range from 1 to 10 wt %.
 11. The testpiece according to claim 1, wherein a proportion of the secondsurfactant in all the components during an enzyme reaction is in a rangefrom 1 to 20 wt %.
 12. The test piece according to claim 1, wherein aweight ratio (A/B) of the first surfactant (A) to the second surfactant(B) is in a range from 1/10 to 1/1.
 13. The test piece according toclaim 1, wherein a reaction pH during an enzyme reaction is adjusted tobe in a range from 6 to
 9. 14. (Added) The test piece according to claim5, wherein the second surfactant is contained in an upper portion of thesample supply layer, the first surfactant and the part of the enzymereagent are contained in a lower portion of the sample supply layer, andthe rest of the enzyme reagent is contained in the detecting layer. 15.(Added) The test piece according to claim 5, wherein cholesterolelastase and cholesterol oxidase are contained in the sample supplylayer as the part of the enzyme reagent, and a peroxidase and asubstrate that develops color by oxidation are contained in thedetecting layer as the rest of the enzyme reagent.
 16. (Added) The testpiece according to claim 10, wherein a proportion of the firstsurfactant in all the components during an enzyme reaction is in a rangefrom 3 to 7 wt %.
 17. (Added) The test piece according to claim 11,wherein a proportion of the second surfactant in all the componentsduring an enzyme reaction is in a range from 7 to 13 wt %.
 18. (Added)The test piece according to claim 12, wherein a weight ratio (A/B) ofthe first surfactant (A) to the second surfactant (B) is in a range from1/3 to 2/3.